Tumor necrosis factor alpha (TNF-.alpha.), also known as cachectin, is a 17 kDa protein produced by neutrophils, activated lymphocytes, macrophages, NK cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells, and some transformed cells. A large number of studies reveal that TNF-.alpha. is produced principally by macrophages and that it may be produced in vitro as well as in vivo. This cytokine mediates a wide variety of biological activities, including: cytotoxic effects against tumor cells, activation of neutrophils, growth proliferation of normal cells, and immunoinflammatory, immunoregulatory, and antiviral responses. TNF-.alpha. also induces the secretion of interleukin-1 (IL-1) and is a primary mediator of inflammation and endotoxin-induced shock. A 26 kDa membrane form of TNF-.alpha. has been described on the surface of monocytes and activated T cells. This molecule may be involved in intracellular communication, as well as cytotoxic activity, and is a surface marker for lymphocyte activation. By a variety of techniques TNF has been shown to exist as a trimer in aqueous solutions; only a small fraction of human TNF molecules occur as monomers at physiological ionic pH.
Two distinct TNF-.alpha. receptors have been identified: a 75 kDa receptor and a 55 kDa receptor, TNFR-.alpha. and TNFR-.beta. respectively. The intracellular domains of the two TNF receptor types are apparently unrelated, suggesting that they employ different signal transduction pathways. While both receptors are capable of binding TNF and activating the transcription factor NFkB, it appears that the expression of each receptor is independently and differentially regulated. Human TNF-.alpha. will bind to both types of receptors with equal affinity on human cells.
TNF has been found to be an important mediator of the pathophysiological effects of a diverse array of invasive diseases, infections, and inflammatory states. As a consequence of its production (or overproduction) in tissues, and the presence of other cytokines in the cellular environment, TNF may ultimately benefit or injure the host. For instance, when produced acutely and released in large quantities into the circulation during a serious bacterial infection, it triggers a state of shock and tissue injury (septic shock syndrome) that carries an extremely high mortality rate (30 to 90%). Three main lines of evidence indicates that TNF plays a central role in the development of septic shock: (1) administration of the cytokine to mammals induces a state of shock and tissue injury that is nearly indistinguishable from septic shock; (2) inhibiting TNF in septic shock prevents the development of both shock and tissue injury and confers a significant survival advantage; and (3) TNF is produced in animals and humans during experimental and clinical septic shock syndrome.
When produced during chronic disease states, TNF mediates cachexia, a syndrome characterized by anorexia, accelerated catabolism, weight loss, anemia, and depletion of body tissues. Weight loss frequently develops during chronic illness and, if not reversed, may kill the host before the underlying disease can be eradicated. For instance, it is not unusual for the patient afflicted with cancer of AIDS to lose 50% of body weight and to succumb to complications of malnutrition. By contrast to starvation, during which protein-conserving adaptive responses are maximally operative, the cachectic host tends to catabolize body energy stores in the face of suppressed intake, thus hastening its own demise.
In addition to septic shock and cachexia, TNF has been implicated in the pathophysiology of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS) and AIDS and has been suggested to perhaps play a role in the development of Alzheimer's disease (AD) and/or the weight loss associated with AD patients.
In rheumatoid arthritis, for instance, there is evidence of macrophage activation with demonstration of increased amounts of two monokines, TNF-.alpha. and IL-1, in the serum but even more in the synovial fluid. TNF-.alpha., an inducer of IL-1, is significantly elevated in rheumatoid arthritis but not in reactive arthritis. Moreover, TNF-.alpha. levels in RA correlate with the synovial fluid leukocyte count and with the ESR (erythrocyte sedimentation rate). TNF is an important mediator of immunity and inflammation and because of its biologic activities (activation of neutrophils, release of arachidonic acid metabolites from synovial cells, induction of cartilage resorption and inhibition of proteoglycan release in cartilage, induction of macrophase chemotactic activating protein ([MCAP]) is one of the potential mediators in chronic arthritis. Studies have shown that monoclonal antibody to TNF can ameliorate joint disease in murine collagen-induced arthritis. In these studies, anti-TNF administered prior to the onset of disease significantly reduced paw swelling and histological severity of arthritis without reducing the incidence of arthritis or the level of circulating anti-type II collagen IgG. More relevant to human disease was the ability of the antibody to reduce the clinical score, paw swelling, and the histological severity of disease even when injected after the onset of clinical arthritis.
More recently, 20 patients with active rheumatoid arthritis were treated with 20 mg/kg of chimeric human/mouse monoclonal anti-TNF-.alpha. in an open phase I/II trial lasting eight weeks. The treatment was well-tolerated and significant improvements were seen in the Ritchie Articular Index, the swollen joint count, and in other major clinical assessments. Significant decreases were seen in serum amyloid A, IL-6 and c-reactive protein.
Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). The majority of infiltrating cells at the site of demyelination are macrophages and T-cells. IL-1 and TNF in the CSF are detected at higher levels and more frequently in patients with active multiple sclerosis than in patients with inactive MS or with other neurological diseases. In a study of MS patients, Beck and colleagues found an increase of TNF and interferon production by peripheral blood mononuclear cells two weeks prior to disease exacerbation. Experimental allergic encephalomyelitis (EAE) is the best characterized demyelinating disease of the CNS in animals. EAE and MS share many characteristics. Ruddle and colleagues used a monoclonal antibody which neutralizes TNF to treat EAE in mice. See Ruddle et al., J. Exp. Med., 1990, 172:1193-1200. The incidence and severity of EAE in the antibody-treated mice were dramatically reduced and the onset of disease was delayed. Moreover, the authors reported that the preventive therapy was long-lived, extending through five months of observation.
TNF-.alpha. levels were measured in serum samples from 73 HIV-1 seropositive patients and in samples from two control groups. All clinical groups of HIV-1-infected patients, regardless of concurrent illness, had significantly elevated levels of both types of soluble TNF receptors (sTNFRs) and immunoreactive TNF-.alpha., with the highest concentrations among the AIDS patients. These TNF parameters were significantly correlated with reduced CD4+ lymphocyte counts. The raised levels of immunoreactive TNF and sTNFRs strongly indicate activation of the TNF-.alpha. system during HIV-1 infection. Levels increase with disease progression and degree of immunodeficiency. Thalidomide, a selective inhibitor of TNF-.alpha. synthesis, has been shown to suppress the activation of latent HIV-1 in a monocytoid (U1) cell line. Associated with HIV-1 inhibition was a reduction in agonist-induced TNF-.alpha. protein and mRNA production. The presence of thalidomide was also shown to inhibit the activation of virus in the peripheral blood mononuclear cells of 16 out of 17 patients with advanced HIV-1 infection and AIDS. A recent study used reverse transcriptase-polymerase chain reaction on homogenized brain tissue to correlate the relative expression of mRNA for TNF-.alpha. with cognitive impairment and with neuropathologic changes in HIV infected patients. Levels of mRNA for TNF-.alpha. from frontal subcortical white matter were significantly greater in patients with HIVD (HIV associated dementia) than in AIDS patients without dementia or in seronegative controls. Elevated levels of mRNA for TNF-.alpha. in HIVD indicate that abnormal cytokine expression may contribute to the pathogenesis of HIVD. Pentoxifylline (PTX), a drug known to block TNF-.alpha. release, was tested in a phase I/II clinical trial of HIV-seropositive patients either alone or in combination with zidovudine (ZDV). The mean HIV-1 viral load, as measured by a quantitative polymerase chain reaction technique, was 1.9-fold above baseline values after 12 weeks of PTX and ZDV compared with 8- to 9-fold greater levels in patients given either agent alone (p&lt;0.05). TNF-.alpha. levels correlated with viral load (p&lt;0.0001) in patients given the combined drug regimen.
Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases of unknown etiology but there is circumstantial evidence that immune mechanisms. play an important role in the pathogenesis of the intestinal lesion and that cytokines produced by lymphoid cells may be critical for the extraintestinal sequelae of the disease. In both Crohn's disease and ulcerative colitis, activation of macrophages seems to be a key feature and increased production of the macrophage-derived cytokines TNF-.alpha., IL-1, and IL-6 have been reported in both diseases. A recent study determined the location and tissue density of cells immunoreactive for TNF-.alpha. in intestinal specimens from 24 patients with chronic inflammatory bowel disease (15 with Crohn's, 9 with ulcerative colitis) and 11 controls (14). There was significantly increased density of TNF-.alpha. immunoreactive cells in the lamina propria of both ulcerative colitis and Crohn's disease specimens suggesting that this degree of TNF-.alpha. production probably contributes significantly to the pathogenesis of both Crohn's disease and ulcerative colitis by impairing the integrity of epithelial and endothelial membranes, increasing inflammatory cell recruitment, and by prothrombotic effects on the vascular endothelium.